Dysregulation of the Immune System in a Natural History Study of 1299 Individuals with Down Syndrome

Dysregulation of the immune system in individuals with Down syndrome is thought to play a major role in the pathophysiology of many clinical presentations. This natural history of disease study took a comprehensive evaluation of the prevalence of different immune related diagnoses in a cohort of 1299 patients with Down syndrome compared to a 2605 control cohort of patients without Down syndrome at Mount Sinai Health System in NY, NY over the past 18 years. We conducted a stepwise analysis of the odds of receiving a diagnosis at the Chapter, Sub-chapter and Diagnosis level of the ICD-CM-10 code system. Individuals in our Down syndrome cohort had higher odds of a diagnosis with inflammatory and autoimmune presentations such as Alopecia areata (OR 6.06, p = 0.01), Other sepsis (OR 4.79, p < 0.001, Purpura and Other hemorrhagic conditions (OR 2.31, p < 0.001), and Rosacea (OR 3.11, p < 0.001). They also presented with lower odds of a diagnosis of Herpesviral infection (OR 0.42, p = 0.01), and Viral warts (OR 0.51, p = 0.04). We posit that dysregulation of the immune system in individuals with Down syndrome has impact on infectious diseases, including lowering the incidence of viral disease, and increasing its severity. Our data also suggests inflammation and autoimmune mediated diseases, in particular of the skin, is exacerbated in individuals with Down syndrome. Finally, there may be a need for greater clinical attention to non-emergent conditions within the Down syndrome patient population as those can also greatly affect quality of life.


Introduction
Down syndrome, or Trisomy 21, is a chromosomal abnormality due to the complete or partial presence of a third copy of chromosome 21 [1].In the United States, it occurs at a rate of roughly 1 in 700-800 live births, with more than 200,000 individuals currently living with Down Syndrome [2][3].Prevalence has increased in the 21st century, largely due to the increased life span of individuals with Down syndrome: in 2010, a mean of 53 years and a median of 58 years as compared to 26 years and 4 years, respectively, in 1950 [4].Much of this trend has been driven by advances in medical and surgical interventions for neonatal and pediatric individuals with Down syndrome.As recently as 1973, epidemiological studies in Sweden and the UK suggested that less than half of individuals born with Down Syndrome were expected to survive the neonatal period; in 2010, the survival rate had increased to 95% [1,5].Down syndrome manifests with signi cant phenotypic variation, although the reasons behind the heterogeneic presentation are elusive.One common hypothesis is that genetic variants in 200-protein coding genes on the extra copy of chromosome 21 may have different cis and trans effects [2,4].
In recent years, this multi-organ multi-system syndrome has sparked a burgeoning literature describing associated comorbidities.Among individuals with Down syndrome, roughly 50% present with congenital heart defects (commonly atrial and ventricular septal defects), more than half present with obstructive sleep apnea, more than half of neonates with Down syndrome present with abnormal thyroid tests, and about 8% of pediatric individuals with Down Syndrome present with epilepsy.Additionally, individuals present with both conductive and sensorineural hearing loss, with ophthalmoplegias (commonly congenital and developmental cataracts and refractive errors), with atlantoaxial instability, with mental health disorders (such as anxiety and depressive disorders), with intellectual disability, and with earlyonset Alzheimer's disease [2,4,6].
The treatment of patients with Down syndrome varies by phenotype and organ system: for example, identi cation of congenital heart defects and gastrointestinal abnormalities should be assessed in the perinatal period, while neurodegenerative disorders and hearing de cits may be screened for throughout the lifetime [2].To guide clinicians, a robust series of international practice guidelines exist for pediatric patients with Down Syndrome for these particular symptoms and signs, in part due to the high historic mortality in the neonatal period, and historically shorter lifespan [6].
More recently, as lifespan has dramatically increased, and early onset diseases have been better managed, it has been recognized that individuals with Down syndrome present with disorders of immune dysregulation [1,7,8].Interestingly they present with both susceptibility to severe infectious diseases (despite having lower incidence of these infections), and concomitant increased levels of autoimmunity and autoin ammation, presenting as alopecia areata, type I diabetes, eczema, atopic diseases of the lung and skin as well as generalized in ammation [8, 9,10,11,12].
Unlike for pediatric patients, no de nitive clinical guidelines currently exist for adults with Down syndrome, especially in the context of immune dysregulation, although some efforts are in progress [2].This may in part contribute to high mortality rates for adult individuals (> 35 years old) with Down syndrome as compared to their peers without Down syndrome.Additionally, many individuals with Down syndrome are at higher risk of chronic disease as they age: nearly 40% of individuals with Down syndrome will develop Alzheimer's disease by the age of 55 [1].
Although some data exists describing the heterogeneous phenotypes and manifestations of Down Syndrome, less is known about how patients with Down syndrome present within the healthcare system across disparate age groups and by organ system or disease grouping.This lack of knowledge presents a roadblock in the development of substantial and effective preventative health guidelines to screen for health issues before they worsen.
Given these gaps in knowledge, we sought to develop a more comprehensive understanding of which disease manifestations are more common in individuals with Down syndrome at a large, urban tertiary/quaternary care center as compared to individuals without Down syndrome.Thus we interrogated the Electronic Health Record to ascertain which diagnoses are more likely in a Down syndrome cohort consisting of 1299 individuals over the last 18 years, as compared to an age, race, sex, and insurancematched control cohort.Our data coincides with the sharp rise in prevalence and life expectancy of patients with Down syndrome and may shed light on how this epidemiological trend manifests in the clinic.Our ndings also add to the growing understanding of the phenotype of the adult patient with Down syndrome and may contribute to the development of a robust and evidence based series of screening guidelines.

Data
We used 18 years of encounter data in the form of International Classi cation of Diseases-10-Clinical Modi cation (ICD-10-CM) codes from the electronic medical record of a large northeastern hospital network including eight hospital campuses and over 410 ambulatory practice locations with over 3,000,000 outpatient encounters and over 130,000 inpatient admissions.Aggregation of the data was performed by the Mount Sinai Data Warehouse using ATLAS, an open-source application developed by the Observational Health Data Sciences and Informatics team [13].Individuals who were diagnosed in any encounter with an ICD-10-CM code were considered positive for that condition in all subsequent analyses.The Institutional Review Board of the Icahn School of Medicine at Mount Sinai approved IRB-18-00638/STUDY-18-00627.
We identi ed 1299 individuals who met the inclusion criteria for the study.

Controls
We matched each individual in our cohort to at least one and a maximum of three age, race, sex, and insurance-matched individuals without Down syndrome.Individuals with < 2 encounters with the health system were excluded from the study.We identi ed 2605 individuals who met the inclusion criteria for controls.

Analysis
We rst analyzed the demographic makeup of our case versus control cohorts by age, sex, race, and insurance status (Table 1).In the cases where the insurance status or race was unknown, we removed the data from the analysis.We calculated p-values and chi squared test statistics for this portion of the analysis.We next analyzed our diagnostic data using the ICD-10 codes in a stepwise manner.We rst analyzed the case versus control data at the ICD-10-CM chapter level (Table 2).In this analysis, any diagnosis within a predetermined range of ICD-10-CM codes was considered a positive hit.Next, we analyzed the data in selected subchapter levels, based on our ndings in the previous step.In this analysis, any diagnosis within a narrower range of ICD-10-CM codes was considered a hit.We selected the three subchapters ("Certain infectious and parasitic diseases", "Diseases of the blood and blood forming organs and certain disorders of the immune process," and "Diseases of the skin and subcutaneous tissue") to investigate further based on relative underreporting of these diagnoses in the literature.We chose not to pursue certain other subchapters because those disease processes are well documented in the literature [15].We next performed an analysis at the diagnosis level based on our previous ndings.In this analysis, a positive hit was determined only when the exact ICD-10-CM code was present in the patient's electronic medical record.Finally, we split our Down syndrome and control cohort into two age bins: >40 years old and 40 + years old to analyze odds of receiving our diagnoses of interest across age groups of the population.We calculated odds ratios, p-values and chi squared test statistics for this portion of the analysis (Fig. 1, 2, 3).
Our control population of 2605 total individuals without Down syndrome was also majority female (57%) and non-Hispanic White (52%).A plurality of the control population was also > 40 years old (38%) and also with roughly equal representation from the 6-

ICD-10-CM Chapter Level
At the ICD-10CM Chapter level we found that individuals in our Down syndrome cohort had lower odds of a diagnosis of "Certain infectious and parasitic diseases" (A00-B99) as compared to control individuals without Down syndrome (Odds Ratio [OR] 0.87, p = 0.06).Individuals in our Down syndrome cohort also had lower odds of a diagnosis of "Neoplasms" (C00-D49) (OR 0.36, p = < 0.001), "Diseases of the skin and subcutaneous tissue" (L00-L99) (OR 0.81, p = 0.01), "Diseases of the musculoskeletal system and connective tissue (M00-M99) (OR 0.68, p < 0.001), "Diseases of the genitourinary system" (N00-N99) (OR 0.73, p < 0.001), and "Factors in uencing health status and contact with health services" (Z00-Z99) (OR 0.64, p < 0.001) (Table 2).These ndings are largely in line with previous studies.Although individuals with Down syndrome are known to be at increased risk of certain neoplasms, this data suggests that overall risk may be decreased.[4,5].

ICD-10-CM Sub-Chapter Level
Within the sub-chapter of "Certain infectious and parasitic diseases" (A00-B99), we found that individuals in our Down syndrome cohort as compared to control individuals without Down syndrome had lower odds of a diagnosis of "Infections with a predominantly sexual mode of transmission" (A50-64) (OR 0.51, p = 0.03).While hard to prove it is reasonable to assume that behavioral aspects of individuals with Down syndrome signi cantly contribute to these outcomes.We also document that "Viral infections characterized by skin and mucus membrane lesions" occur at lower rates (B00-B09) (OR 0.42, p < 0.001).
However, they also had higher odds of a diagnosis of "Other bacterial diseases" (A30-49) (OR 3.06, p < 0.001) (Fig. 1) Within the sub-chapter "Diseases of the skin and subcutaneous tissue" (L00-L99), we found that individuals in our Down syndrome cohort as compared to control individuals without Down syndrome had lower odds of a diagnosis of "Radiation related disorders of the skin and subcutaneous tissue (L55-L59) (OR 0.27, p = 0.03) and "Urticaria and erythema" (L49-54) (OR 0.56, p = 0.02).However individuals with Down Syndrome had higher odds of a diagnosis of "Infections of the skin and subcutaneous tissue" (L00-L08) (OR 1.32, P = 0.03) and higher odds of a diagnosis of "Disorders of skin appendages" (L60-75) (OR 1.33, P = 0.01) (Fig. 1).To which extent the diagnoses reported as rarer are truly so in individuals with Down Syndrome is hard to ascertain as it is possible they are simply underreported by individuals themselves and underdiagnosed by the providers.In this sub-chapter, certain diagnoses may be driven by environmental versus immune factors and require further granular investigation to untangle.
Within the sub-chapter "Diseases of the blood and blood forming organs and certain disorders of the immune process" (D50-D89), we found that individuals in our Down syndrome control as compared to control individuals without Down Syndrome had lower odds of a diagnosis of "Nutritional anemias" (D50-D53) (OR 0.59, p = 0.01).However they had higher odds of a diagnosis of "Other disorders of blood and blood forming organs" (D70-77) (OR 2.91, p < 0.001) (Fig. 1).Here again, the interplay between caretakers of individuals with Down syndrome, the Down syndrome phenotype, and the medical system may be protective in certain instances such as dietary intake.

Bacterial and Viral Infections
We found that individuals in our Down syndrome cohort as compared to control individuals without Down syndrome had lower odds of a diagnosis "Herpesviral infections" (B00) (OR 0.42, p = 0.01), "Viral warts" (B07) (OR 0.51, p = 0.04), and "Unspeci ed viral infections characterized by skin and mucous membrane lesions" (OR 0.42, p = 0.01) (Fig. 2).The extent to which this is mediated by additional copies of type 1 interferon receptor (IFNAR) encoded by chromosome 21 contributing to a hyperactive response to key antiviral cytokine type I interferon is worth exploring further.(36813963) (32572726) (27472900) Interestingly individuals with Down syndrome had higher odds of a diagnosis of "Other sepsis" (A41), (OR 4.79, p < 0.001) (Fig. 2), which is perhaps founded in their already increased steady state of in ammation as documented by several studies [8,16,17,18].

Blood and Blood Forming Organs and Diseases of Immune Mechanism
We found that individuals in our Down syndrome cohort as compared to control individuals without Down syndrome had lower odds of a diagnosis of "Iron de ciency anemia" (D50) (OR 0.47, p < 0.001).However, they had higher odds of a diagnosis of "Other nutritional anemias" (D53) (OR 6.04, 0.03), "Purpura and other hemorrhagic conditions" (D69) (OR 2.31,, p < 0.001), "Other disorders of white blood cells" (D72) (OR 2.18, p = 0.000), and "Other and unspeci ed diseases of blood and blood forming organs" (D75) (OR 6.50,, p < 0.001) (Fig. 2).Previous studies on hematologic abnormalities in individuals with Down syndrome have largely focused on malignancies and the creation of suggested screening guidelines [2,5].This nding sheds light on the consequences of immune dysregulation with the hematologic system of individuals with Down syndrome, which has been less well studied.

Skin and Subcutaneous Tissue
We found that individuals in our Down syndrome cohort as compared to control individuals without Down syndrome had lower odds of a diagnosis of "Urticaria" (L50) (OR 0.41, p = 0.03), "Androgenic Alopecia" (L64) (OR 0.08, p = 0.08), and "Acne" (L70) (OR 0.66, p = 0.04).However, they had higher odds of a diagnosis of " Alopecia Areata" (L63) (OR 6.06, p = 0.01), "Rosacea" (L71) (OR 3.11, p < 0.001), and "Other follicular disorders" (L73) (OR 3.70, p < 0.001) (Fig. 2).One possible explanation for these skin related disorders can be founded in the increased levels of steady state in ammation which leads to hyperactive immune system and ultimately autoimmunity, which can be both T and B cell governed [8, 11,12].Only detailed molecular understanding of the rogue immune system can help us rst understand pathophysiology of particular diseases, and then allow us to deploy appropriate therapeutic modalities.

Age Binned Analysis
There were 2496 individuals in our < 40 year old cohort (case n = 872, control n = 1624) and 1408 individuals in our 40 + year old cohort (case n = 427 control n = 981).We found that the odds of a diagnosis of "Other sepsis" (A41) in our Down syndrome cohort as compared to control individuals was increased in the < 40 year old age bin (OR 16.6, p < 0.001) as compared to the 40 + year old age bin (OR 3.67, p < 0.001).Down syndrome individuals in the < 40 year old age bin also had higher odds of receiving

Discussion
We identi ed important differences in the odds of receiving certain diagnoses between our cohort of 1299 individuals with Down syndrome and our control cohort of 2605 individuals without Down syndrome.Previous studies have used analytic methods such as grouping diagnoses of interest for analysis [15].We employed a novel stepwise approach in our three phase analysis across ICD-10-CM codes at the chapter, subchapter, and diagnostic level in order to avoid selection bias.Using this method, we were able to nd the main drivers of protective and risk factors within the disease groupings and organ systems we identi ed.Our results shed light on the complex interplay between genetics, pathology, and suggests the need for further study into the role of caretakers in the non-emergent health of individuals with Down syndrome.
Individuals with Down syndrome were overall less likely to receive a diagnosis within the chapter of "Certain infectious and parasitic diseases" as previously noted [9].This protection may be explained by the inclusion in this category of common STIs such as "Herpesviral infections" (OR 0.42, p = 0.01) and "Viral warts" (OR 0.51, p = 0.04).Explanations for this phenomenon in the literature with regards to STIs include potentially delayed onset of sexual maturity in the Down syndrome population, reliance on caregivers to notice signs like warts, and insu cient data on sexual activity in the Down syndrome population [15].Equal weight in the case of protection against other viral etiologies of infection must be given to the growing understanding of IFNAR in uence in the Down syndrome phenotype.Previous work has shown that individuals with Down syndrome may have initial resistance to viral infection due to gene dosage effect of IFNAR1 and IFNAR2, but more serious sequelae to infection once it occurs due to excessive negative regulation of type I IFN, via USP18 [9].Our analysis additionally shows that individuals were signi cantly more likely (OR 4.79, p < 0.001) to receive a diagnosis of "Other sepsis," which includes sepsis due to Staphylococcus aureus, Escherichia coli, and other common bacterial ora.
Others have documented large levels of in ammation at baseline in individuals with DS, and perhaps during bacterial infections this may yield a more fertile ground for serious complications like sepsis.
Individuals with Down syndrome were overall less likely to receive a diagnosis within the chapter "Diseases of the skin and subcutaneous tissue."However, after our stepwise approach to analysis, we found that they had signi cantly higher odds of receiving a diagnosis of "Alopecia Areata," "Rosacea," and "Other follicular disorders" (a category that includes Hidradenitis Suppurutiva) (OR 6.06, p = 0.01, OR 3.11, p < 0.001, OR 3.70, p < 0.001, respectively).However they had reduced odds of a diagnosis of "Androgenic Alopecia," with none of our cohort of individuals with Down syndrome receiving a diagnosis, and "Acne" (OR 0.66, p = 0.04).This may be a function of the role that family members, home health aides, and other caretakers play in the medical management of individuals with Down syndrome.
"Androgenic Alopecia" and "Acne" may be more likely to be discussed during clinical encounters by individuals who are concerned with cosmetic aspects of their health, while "Alopecia Areata" and "Rosacea" may be more concerning to a secondary observer.The pathophysiologies of "Alopecia Areata," "Rosacea," and follicular disorders such as Hidradenitis Suppurutiva are less well understood, but are likely to be immune in nature.The association of increased odds of these disorders in our cohort of individuals with Down syndrome, with presumed immune dysregulation as a driving factor, further reinforces these hypotheses.
Individuals with Down syndrome were overall more likely to receive a diagnosis within the chapter "Diseases of the blood and blood forming organs and certain disorders involving the immune mechanism."However, after our stepwise analysis we found that they had signi cantly lower odds of receiving a diagnosis of "Iron de ciency anemia" (OR 0.47, p < 0.001).A common presenting symptom of iron de ciency anemia is fatigue.The reduced odds in this case may be due inability to communicate symptoms.Individuals with Down syndrome were more likely to have higher odds of a diagnosis of "Other nutritional anemias, "Purpura and other hemorrhagic conditions," "Other disorders of white blood cells," and "Other and unspeci ed diseases of blood and blood forming organs."Certain pathologies such as Immune thrombocytopenic purpura (ITP) and Drug rash with eosinophilia and systemic symptoms (DRESS syndrome) are included in the latter two diagnostic categories, which are known to be conditions mediated by a hyperactive immune system.Overall, these ndings may be due to more extensive routine laboratory workups for individuals with Down syndrome or previously unexplored downstream effects of the immune dysregulation present in Down syndrome.
In our age binned analysis, younger individuals with Down Syndrome compared to their controls had higher odds of diagnoses of "Other sepsis" (A41), "Rosacea" (L71), "Other follicular disorders" (L73), and "Other disorders of white blood cells" (D72) than older individuals with Down syndrome compared to their own controls.This may be a result of younger individuals with Down syndrome taking more autonomy over their own healthcare, greater clinical attention in recent years to the broad spectrum of pathologies in individuals with Down syndrome, or simply signi cantly earlier onset of these signs and symptoms compared to age matched groups.Our analysis cannot assess causality but we suggest that this is fruitful ground for further mixed methods analyses of how younger patients with Down syndrome and their caregivers present within the healthcare system as opposed to their older peers.
Limitations of this study included our stepwise approach.We initially set out to investigate how individuals with Down syndrome are diagnosed within the con nes of the ICD-10-CM coding system as compared to their peers without Down syndrome.There are over 70,000 codes within the system.By beginning at the chapter level, we may have obscured or glossed over diagnoses with higher prevalence at certain subchapter, sub-subchapter, or diagnostic levels.This top-down approach, as opposed to a more targeted approach, may have uncovered certain incidental ndings within the ICD-10-CM coding system that are less relevant on the clinical level.Future studies should consider bracketing analysis of these datasets by decade to account for changes in medical management and a growing understand of the phenotype of the individual with Down syndrome.
In summary, given the data on 1299 individuals with Down syndrome in our health system we posit that dysregulation of the immune system in individuals with Down syndrome has impact on infectious diseases, both lowering the incidence of viral disease, but increasing its severity, likely by molecular mechanism governing regulation of type I IFN response.Akin to this we posit that documented hyperin ammatory state leads to a milieu where sepsis is more likely to occur upon bacterial infections.
Our data also suggests in ammation and autoimmune mediated diseases of the skin is exacerbated in individuals with Down syndrome.We also suggest that there is a need for greater clinical attention to non-emergent conditions within the Down syndrome patient population.Finally, we hypothesize that the increased odds of certain diagnoses within a younger Down syndrome cohort may be due to increased in ammation or a combination of other unknown factors.Deeper molecular and biochemical evaluation of the underlying immune regulation will shed deeper light on pathophysiology and allow us to deploy many a drug in the growing arsenal of anti-in ammatories in this population.

Declarations
Figures

Table 2
Odds of Receiving a Diagnosis Within ICD-10-CM Chapter Compared with Control The majority of the control population insurance status was private insurance or Medicaid (40% and 35%, respectively) with individuals insured by Medicare representing 24% of the population.The case and control populations were not signi cantly different by sex, age, or race.The cohorts were signi cantly different by insurance status (p = 0.002, with Pearson's chi squared test = 18.29).
Some of these changes in prevalence of diagnoses may be a re ection of the concept of in ammation related aging in Down syndrome.